Social stimulation and the resumption of copulation in Rhesus (Macaca mulatto) and stumptail (Macaca arctoides) Macaques

Copulatory data derived from observations of social groups of rhesus and stumptail macaques were analyzed to test the hypothesis that pairs of animals would resume copulation significantly sooner if a second male copulated with the female shortly after the first male’s ejaculation. Data from both groups supported the hypothesis. These results, extending previous studies in Macaca nemestrina,suggest that the shortening of copulatory intervals by social stimuli occurs in several species, both in social groups and in experimentally created triads. These findings also are consistent with the hypothesis that socially mediated resumption of mating is related to intrasexual competition among males.     

Analysis of the mating behavior and some possible causes of male copulatory success in Dryas iulia alcionea (Lepidoptera, Nymphalidae, Heliconiinae)

In this study we examined the mating behavior of Dryas iulia and the acceptance and rejection mechanisms of females during courtship activity. An ethogram of mating behavior was organized on the basis of 100 h of observation in an insectarium. Several different behaviors were catalogued and separated into two behavioral repertoires (pre-coupling, post-coupling). The behavioral sequence of mating behavior was also analyzed using a total of 53 pairs of D. iulia. The courtship activities involved interactions between the sexes in three sequential phases—the aerial, air-ground, and ground phases. In 49% of observations the courtship activities led to copulation. The mean time to the occurrence of the first interaction between males and females did not differ between interactions with and without copulation. The behavior of females and males in the two groups were analyzed and differences were found between behavioral activities of both sexes. The results indicate that males’ insistence on courtship does not affect their copulatory success, and that females play a decisive role in copulation occurrence. Analysis of behavioral transitions showed there are many alternative behavioral routines in interactions with and without copulation. The number of behavioral transitions recorded was smaller in the group in which copulation occurred, indicating that males with copulatory success modified their behavior less frequently. Successful males recorded more transitions with a probability of occurrence greater than 0.4, and their behavioral activity was also less reticulated. Analysis using the stereotyping index showed that situations in which copulations occurred were more stereotyped than those in which it did not.     

Social dominance and female reproductive behaviour in rhesus monkeys (Macaca mulatta)

The relationship between dominance rank and female sexual behaviour was examined in rheusus monkeys (Macaca mulatta) living in a social group. High-ranking females engaged in copulatory series as frequently as lower-ranking females. Furthermore, lower-ranking females copulated with as many available males as did high-ranking females. Social rank did appear to influence the pattern of sexual activity exhibited, in that copulatory series were more often initiated by the higher-ranking animal of the mating pair. Copulatory series involving high-ranking females were characterized by more mounts by males and were longer in duration. Higher-ranking animals interfered more often with copulatory series involving other animals, but such interference was not effective in preventing completion of the series. These data indicate that any reproductive advantage conferred to high-ranking females is not the result of sexual competition in social-living rhesus monkeys.     

EVIDENCE FOR WIDESPREAD COURTSHIP DURING COPULATION IN 131 SPECIES OF INSECTS AND SPIDERS,AND IMPLICATIONS FOR CRYPTIC FEMALE CHOICE

Male courtship behavior is generally thought to function prior to copulation, as an inducement to the female to allow the male to copulate with her; this study indicates however, that male courtship during and following copulation (“copulatory courtship”) is common in insects and spiders (81% of 131 species in 102 genera and 49 families, mostly Coleoptera, Hemiptera, Diptera, and Araneioidea). Copulatory courtship is apparently evolutionarily labile, as expected if it is under sexual selection; intrageneric variation occurred in all 17 genera in which more than one species was observed. In 81% of 94 species with copulatory courtship, the male abandoned the female soon after copulation ended; thus, copulatory courtship appears not to function generally to induce acceptance of further copulatory attempts. The most likely explanation for copulatory courtship is that it represents attempts by males to influence cryptic female choice. This suggests that an aspect of sexual selection by female choice not considered by Darwin may be more important than previously appreciated and that the common practice in evolutionary studies of measuring male reproductive success by counting numbers of copulations may sometimes be misleading because of cryptic female choice during and after copulation.     

Histofluorescent labelling of catecholaminergic structures in rotifers (Aschelminthes)

Summary 1. Catecholaminergic structures in the male Brachionus plicatilis were investigated, using aqueous dansylpropranolol as fluorescent label of neurotransmitter receptors. 2. All major organs of the male are innervated by catecholaminergic systems, that may also be involved in the regulation of copulatory behavior. 3. Cryostat-sectioned preparations of the female B. plicatilis were also investigated. They provided additional information to findings on whole animals reported in our previous paper (Keshmirian and Nogrady 1987a).     

Reproductive behavior of the captive Sunda pangolin (Manis javanica Desmarest, 1822)

Understanding reproductive behavior is important for the conservation of endangered species, but research on the reproductive behavior of Sunda pangolins (Manis javanica Desmarest, 1822) is still very scarce. In this study, we used focal animal sampling and all-occurrence recording by an infrared monitor to observe the behaviors of two Sunda pangolins during a 5-day breeding period at the Pangolin Research Base for Artificial Rescue and Conservation Breeding of South China Normal University (PRB-SCNU). The behavioral characteristics and breeding strategies were analyzed, and the results were discussed together with information on other captive Sunda pangolin pairs at PRB-SCNU. The results found that there was no obvious estrus behavior in the captive female, while the male could exhibit sexual excitement and courtship behavior after a brief introduction period. Repeated copulation continued over many days after the female accepted the courtship. The average duration of copulatory behavior was 248.9 ± 148.7 s (n = 25), and all copulation occurred between 20:00 and 08:00 hr in the natural day–night photoperiod. The mating position of Sunda pangolins was lateral–ventral and was classified as the ninth or eleventh pattern under both Dewsbury's and Dixson's classification systems. This study can provide scientific guidance for the captive breeding and management of Sunda pangolins and other pangolin species, which is of great significance for ex situ conservation tactics.     

Conspecificity of Elachista nigra and Elachista orbicularis (Elachistaceae, Phaeophyceae)

A taxonomic study of two brown algal species, Elachista nigra Takamatsu and Elachista orbicularis (Ohta) Skinner (Elachistaceae), was performed on the basis of morphological observations of field‐collected and laboratory cultured specimens from Japan (including their type localities) and molecular phylogenetic analyses. The two species had been distinguished by developmental patterns of paraphysis‐ and plurizoidangium‐bearing erect filaments, such filaments of E. nigra developing from wide erect filaments and those of E. orbicularis developing directly from basal prostrate filaments. However, many specimens investigated in the present study showed forms intermediate between these two patterns. Molecular phylogenetic analyses (including five additional elachistacean species) based on the internal transcribed spacer (ITS)2 region of the nuclear ribosomal RNA (nrRNA) gene showed a close relationship between all samples of E. nigra and E. orbicularis, and that the developmental patterns of paraphysis‐ and plurizoidangium‐bearing erect filaments were homoplasious. On the basis of these morphological and molecular data, E. orbicularis was reduced to synonymy with E. nigra. The ITS2 sequences of E. nigra were significantly different between samples from the Sea of Japan and those from the Pacific Ocean with several insertion/deletion and substitution mutations.     

Social–sexual behavior seasonality in captive beluga whales (Delphinapterus leucas)

Observation of wild beluga whales (Delphinapterus leucas) indicates that this species breeds seasonally, and hormonal assays of captive animals show corresponding fluctuations in reproductive hormones. The question remains, however, whether copulatory behavior itself occurs seasonally. As an index of copulatory behavior, data are reported here on the frequency with which pelvic thrusting occurs over the calendar year. Male-on-female thrusting did vary significantly across months, with a clear peak in activity in March. On the other hand, male-on-male pelvic thrusting did not differ significantly across months. Possible functional roles are suggested for this latter enigmatic behavior.     

Phylogeny and biogeography of the scaleless scale worm Pisione (Sigalionidae,Annelida)

Pisione is a scaleless group of small scale worms inhabiting sandy bottoms in shallow marine waters. This group was once considered rare, but now 45 described species can be characterized, among others, by their paired, segmental copulatory organs (one to multiple external pairs), which display a complexity of various accessory structures. The evolutionary significance of these unique organs was suggested in the late 1960s, but has been heavily debated since the late 1990s and remains controversial. In the present paper, we study the internal relationships within Pisione, employing combined phylogenetic analyses of both molecular and morphological data from 16 terminals of Pisione, as well as two terminals of Pisionidens, and eight additional scale worms as outgroups. Our taxon sampling covers all geographical areas where the genus has been reported, as well as most of their morphological and copulatory variability, including representatives of the “africana,” “remota,” “crassa,” and “papuensis” groups, established previously by Yamanishi. We hereby provide a first insight into the relationships of the genus, testing previously proposed hypotheses on the evolutionary significance of male copulatory structures within Pisione, while attempting to understand patterns of distribution. The phylogenetic analyses using maximum likelihood and Bayesian methods consistently recovered two large clades spanning the East Atlantic (including the Mediterranean) and the Indo‐Pacific–West Atlantic, respectively. Character optimization on our trees revealed a high degree of homoplasy in both non‐reproductive and sexual characters of Pisione, with buccal acicula found to be the sole apomorphy among the morphological features assessed herein, with none defining the biogeographical subclades within. Overall, our comparative analyses highlight the high degree of morphological variation in this widely distributed genus, rejecting previous assertions of an increasing number and complexity of copulatory structures across the genus.     

Temperature-related breakdowns in the coordination of mating in Enchenopa binotata treehoppers (Hemiptera: Membracidae)

Temperature influences the expression of a wide range of behavioral traits in ectotherms, including many involved in the initiation of pair formation and mating. Although opportunities to mate are thought to be greatest when male and female activity overlap, sex-specific behaviors and physiology could result in mismatched thermal optima for male and female courtship. Here, we investigate how conflicts in the thermal sensitivity of male and female courtship activity affect patterns of mating across temperatures in Enchenopa binotata treehoppers (Hemiptera: Membracidae). These plant-feeding insects coordinate mating with plant-borne vibrational signals exchanged in male–female duets prior to pair formation. We manipulated temperature across an ecologically relevant range (18–36ºC) and tested the likelihood of individual male and female E. binotata to engage in courtship activity using vibrational playbacks. We then staged male–female mating interactions across the same temperature range and quantified the thermal sensitivity of mating-related behaviors across stages of mating. Specifically, we measured the timing of duetting, the likelihood for key pre-copulatory behaviors to occur, whether the pair mated, and copulation duration. We found sex-specific thermal sensitivity in courtship activity: Males showed a clear peak of activity at intermediate temperatures (27–30ºC), while females showed highest activity at the hotter thermal extreme. Mating rates, courtship duets, and copulatory attempts were less likely to occur at thermal extremes. Also, duetting occurred earlier and copulation was shortest at higher temperatures. Overall, our data suggest that sexes differ in how temperature affects mating-related activity and some processes involved in mate coordination may be more sensitive than others across variable thermal environments.     

Evidence for mouse sulfhydryl oxidase‐assisted cross‐linking of major seminal vesicle proteins

Copulatory plug formation in animals is a general phenomenon by which competition is reduced among rival males. In mouse, the copulatory plug formation results from the coagulation of highly viscous seminal vesicle secretion (SVS) that is rich in proteins, such as dimers of SVS I, SVS I + II + III, and SVS II. These high‐molecular‐weight complexes (HMWCs) are also reported to be the bulk of proteins in the copulatory plug of the female mouse following copulation. In addition, mouse SVS contributes to the existence of sulfhydryl oxidase (Sox), which mediates the disulfide bond formation between cysteine residues. In this study, flavin adenine dinucleotide (FAD)‐dependent Sox was purified from mouse SVS using ion exchange and high‐performance liquid chromatography. The purified enzyme was identified to be Sox, based on western blot analysis with Sox antiserum and its capability of oxidizing dithiothreitol as substrate. The pH optima and thermal stability of the enzyme were determined. Among the metal ions tested, zinc showed an inhibitory effect on Sox activity. A prosthetic group of the enzyme was identified as FAD. The K_m and V_max of the enzyme was also determined. In addition to purification and biochemical characterization of seminal vesicle Sox, the major breakthrough of this study was proving its cross‐linking activity among SVS I–III monomers to form HMWCs in SVS.     

Inhibition of the growth and development of mosquito larvae of Culex pipiens L. (Diptera: Culicidae) treated with extract from flower of Matricaria chamomilla (Asteraceae)

Mosquitoes can transfer many adverse diseases to human and animals therefore, there is a need to fight their spread. Among promising larvicidal sources is the use of plant extracts, which will play an important role in the future. This study was conducted to assess the larvicidal activity of Ferula hermonis Boiss,Achillea millefolium, Salvia officnalis, Psidium guaja and Matricaria chamomilla extract against Culex pipiens. The plant materials were extracted with hexane, dichloromethane, ethyl acetate, and methanol using a Soxhlet extractor. The extracts were evaluated for larvicidal activity against Cx. pipiens the mortality was monitored after 24 and 48 h of exposure. None of the extracts tested showed larvicidal acitivties except M. chamomilla. The ethyl acetate extract showed the most promising larvicidal activity with LC₅₀ values of 287.1 and 209.4 ppm after 24 and 48 h of exposure, respectively. Treatment of the eggs with different concentrations of the active extract decreased the hatchability of the eggs dose dependently from 95 to 86.49%. Similarly, the pupal duration increased in treated groups. The larval period lasted for 12 d, whereas that of the control group lasted for 10 d. Furthermore, the pupal period lasted 3 d (control 2 d) in treated groups. The data also revealed a significant decrease in the growth index in treated groups (0.00–7.53) than that of the control (8.5). The GC–MS analysis revealed the presence of 1.6.10‐dodecatriene, 7,11‐dimethyl‐3‐methylene (89.68%), 1,6‐cyclodecadiene, 1‐methyl‐5‐methylene‐8‐(1‐methylethyl)‐, [S‐(E,E)] (6.34%), 2H‐pyran ?3‐ol (4.04%), and 2H‐1‐benzopyran‐2‐one (0.079%).     

Immunochemical studies of the combining sites of the two isolectins, A4 and B4, isolated from Bandeiraea simplicifolia

The tissue distribution and the effects of starvation and streptozotocin-induced diabetes on insulin B chain-degrading neutral peptidase activity in the rat have been studied. The neutral peptidase activity in tissue extracts was determined by measuring the formation of trichloroacetic acid-soluble radioactivity from ¹²⁵I-labeled B chain of insulin in 0.1 m Tris buffer (pH 7.2). Inhibition by several different compounds (EDTA, dithiothreitol, and potassium phosphate) which are known to inhibit the purified enzyme and the effects of pH suggest that the B chain-degrading activity measured in each of 12 tissue extracts may be similar to the neutral peptidase recently purified from rat kidney (P. T. Varandani and L. A. Shroyer, 1977, Arch. Biochem. Biophys., 181, 82–93). Neutral peptidase activity was observed in all tissues examined and varied in the order kidney ? intestine > pancreas, testis > liver > thymus > heart, skeletal muscle, diaphragm > lung, spleen > fat. Neutral peptidase activity in kidney, liver, fat, and skeletal muscle from diabetic animals was significantly depressed when compared with the levels in these tissues from normal animals. Insulin treatment of diabetic animals raised the neutral peptidase activity in kidney, liver, and fat to levels equivalent to or even exceeding normal levels; however, activity in skeletal muscle persisted at depressed levels. Heart muscle neutral peptidase activity was not significantly affected in either diabetes or starvation. In the liver, starvation reduced the level of neutral peptidase activity while subsequent refeeding raised the activity to a level exceeding the control. Opposite effects were observed in kidney: starvation increased neutral peptidase activity while refeeding brought the activity back to normal levels. Only small decreases in neutral peptidase activity were observed in fat and skeletal muscle after 24 h starvation, but were not evident after 64 h starvation. The changes in neutral peptidase activity correlated well with the changes in glutathione-insulin transhydrogenase activity previously reported in liver and kidney.     

Selected plasma biochemistry parameters in gilthead seabream (Sparus aurata) juveniles

The aim of this study was to assess plasma biochemistry parameters with the potential of being used as indicators of the nutritional status for healthy gilthead seabream juveniles. Triplicate groups of 18 seabream (body weight of 58 g) were kept unfed for 24 h, 7 or 14 days. Nine fish per treatment were then sampled randomly for blood collection and the following parameters analyzed in the plasma using standard clinical methods: glucose; protein; triglycerides; cholesterol; calcium; magnesium; inorganic phosphorus; alkaline phosphatase (ALP); aspartate aminotransferase (AST); lactate dehydrogenase (LDH); gamma‐glutamyl transferase (GGT); creatine phosphokinase (CPK); and lipase. Biochemical parameters showed lower variability among individuals than did enzymatic parameters. Plasma glucose, protein, cholesterol, calcium and inorganic phosphorus levels were inversely related to the duration of starvation. On the contrary, plasma triglycerides decreased significantly during the first week of starvation and remained stable in the second week. Plasma ALP, AST and LDH decreased significantly after 1 week of starvation and then remained constant. In healthy seabream juveniles, plasma glucose, protein, cholesterol, calcium and inorganic phosphorus are responsive to starvation and may be useful indicators of the nutritional status of the animals. Indicative baseline reference values for gilthead seabream juveniles starved for 24 h and held at optimum temperature are: protein, 3.7–4.9 g dl^?1; cholesterol, 341–407 mg dl^?1; calcium, 13.1–8.0 mg dl^?1; and inorganic phosphorus, 10–14.2 mg dl^?1. Plasma triglycerides, along with plasma enzyme activities, may be useful as indicators of short term starvation. For these parameters baseline values after 1 week of starvation were: triglycerides: 138–230 mg dl^?1; ALP: 58–125 U L^?1; AST: 15–127 U L^?1; and LDH 61–677 U L^?1. Plasma glucose is only responsive to longer starvation periods, remaining relatively stable during the first week of starvation, and ranging from 59 to 196 mg dl^?1.     

Copulatory behavior of adult tamarins (Saguinus fuscicollis) castrated as neonates or juveniles: Effect of testosterone treatment

The sexual interactions of Saguinus fuscicollis males castrated as neonates, at 37 days of age, or prepubertally with adult intact females were studied. Prepubertally castrated males were observed while receiving testosterone, and while being treated with saline. Males castrated neonatally or at 37 days of age were observed while receiving testosterone. Neonatal castrates had previously been studied without hormone treatment and therefore no control condition was included for these animals. Prepubertally castrated males showed Mounts, Mounts with Thrusts, and Sexual Tongue Flicking when treated with saline only. In three of the four males, all measures of sexual behavior increased with testosterone treatment. Neonatally castrated males had failed to display any mounting or thrusting without testosterone treatment during a previous study. During the present study, three of the four males did not respond to testosterone treatment with sexual behavior. The fourth male and one male castrated at 37 days of age displayed some sexual behavior. These results suggest that most neonatally castrated males are not able to respond to testosterone with the activation of copulatory behavior. The findings are consistent with the hypothesis that in callitrichids the sensitive period for behavioral differentiation is shifted into neonatal life. However, some neonatally castrated males show a weak response to testosterone. This may reflect an extended and perhaps partially prenatal period of sensitivity.     

Copulatory Mechanism in Holocnemus pluchei and Pholcus opilionoides,With Notes on Male Cheliceral Apophyses and Stridulatory Organs in Pholcidae (Araneae)

Huber, B. A. 1994. Copulatory mechanism in Holocnemus pluchei and Pholcus opilionoides, with notes on male cheliceral apophyses and stridulatory organs in Pholcidae (Araneae).—Acta Zoologica (Stockholm) 76: 291–300. The pholcid spiders Holocnemus pluchei (Scopoli, 1763) and Pholcus opilionoides (Schrank, 1781) are investigated with respect to functional morphology of their genital organs using freeze-fixation of spiders during copula in liquid nitrogen and subsequent preparation of histological serial sections of the copulatory organs in functional contact. Special attention is paid to the mode of male pedipalpal arrestation before copulation, which is achieved in two quite different ways: in Pholcus by contact of the lateral cheliceral apophysis with the pedipalpal trochanter-apophysis, in Holocnemus by locking the pedipalpal trochanter between chelicera and pedipalpal coxa. The condition in Pholcus is considered to be apomorphic and to present a synapomorphy of about a dozen genera for which the name “Pholcus-group” is proposed. The stridulatory apparatus of Holocnemus pluchei is described, its biological significance discussed and an overview of accounts on stridulation in Pholcidae given.     

Towards a characterization of the locomotor activity rhythm of the desertic species Porcellio olivieri (Crustacea,Isopoda) from Gabes (South of Tunisia)

The locomotor activity rhythm of the isopod, Porcellio olivieri, was investigated in Gannouch site in the south of Tunisia. The rhythm was monitored under constant temperature in individual animals in winter under two simultaneous regimens: the light–dark (LD) cycle and the continuous darkness (DD). Results revealed that whatever regimens, actograms, and mean activity curves showed that specimens of P. olivieri concentrated their activity during the experimental and subjective night. The species exhibited a locomotor rhythm period significantly shorter under LD (T = 23h13 ± 0h44) than DD (τ = 24h28 ± 0h58). However, the locomotor activity rhythm was less stable and the individuals were significantly more active under entraining conditions than constant darkness. The activity pattern of this species will be discussed as an adaptative strategy to respond to environmental conditions.     

Effect of meteorological variates on persistence of primary conidia and capilliconidia of Erynia radicans (Zygomycotin: ntomophthorales) under natural conditions

Persistence of conidia of an isolate of Erynia radicans (Syn. Zoophthora radicans) was investigated in relation to the meteorological conditions which occurred during autumn-winter of 1990–91 in the coastal plain in Israel. Capilljconidia shielded from the sun, placed on the abaxial surface of leaves of Plumeria acuminata, persisted for 24 h to at least 120 h. Exposed capilliconidia, placed on the adaxial surface of the same leaves, died within 24 h. Almost all the primary conidia shielded from the sun (placed on the abaxial surface of the same leaves) died within a single day. Conidial viability was expressed in subsequent germination on an agar medium. Capilliconidial persistence was closely related to the daily air temperatures, expressed as cumulative day-degrees. Differences in relative humidity had no substantial effect on capilliconidial mortality. At daytime temperatures of ≤ 20°C, mortality after 24 h incubation was lowest (≤ 34%) and the persistence duration, longest (at least 120 h). Increases in daytime temperature up to 24°C for a few hours increased mortality (37–57% after 24 h incubation) and shortened the persistence duration (72–120 h). Exposure to 24–29°C during daytime greatly increased mortality (65–58% after 24 h) and further shortened the persistence duration (24–48 h). Daytime temperatures of > 29°C were lethal to all capilliconidia within 24 h. Temperature had a profound effect on capilliconidial persistence also under controlled environmental conditions. The significance of capiliiconidial persistence is discussed in relation to activity of the fungus in its natural environment.     

Endogenous activity rhythm in Talitrus saltator,Britorchestia brito (Crustacea,Amphipoda) and Tylos europaeus (Crustacea,Isopoda) from Barkoukech Beach (Tabarka,Tunisia)

The locomotor activity rhythm of three supralittoral crustacean species, Tylos europaeus (terrestrial isopod) and Britorchestia brito and Talitrus saltator (amphipod, Talitridea), was investigated in Barkoukech Beach. The rhythm was monitored in individual animals in spring under continuous darkness and constant temperature. Results revealed that whatever the species, actograms and mean activity curves showed that specimens of T. europaeus and T. saltator concentrated their activity during the subjective night; whereas, B. brito concentrated its activity during the subjective day. The three species exhibited a circadian locomotor rhythm with mean circadian period, respectively, equal to 24h41′ ± 0h44′, 24h46′ ± 0h55′ and 25h21′ ± 1h16′. The most stable locomotor rhythm was found in T. saltator (0.444 ± 0.246). Furthermore, individuals of T. europaeus were significantly more active (10h47′ ± 2h52′) than the two amphipods. Differences in the activity patterns of the three species will be discussed as an adaptative strategy to respond to interspecific competitive interactions.     

Some effects of early starvation on the survival and development of barnacle nauplii,Balanus improvisus (Darwin)

Newly hatched stage I–II nauplii of Balanus improvisus (Darwin) were “totally starved” (until death) or “partially starved” for the first 48 h and 96 h of their development. Daily mortality and molting were monitored throughout larval development in both starved and fed control groups. Fed control animals exhibited a largely synchronous molting pattern with instars of equal duration. Total starvation suppressed molting beyond stage II; 50% mortality occurred in ≈4 days at both 15 and 21 °C, while longest survival time was 7 days at 15 °C and 6 days at 21 °C. At 15 °C, partially starved nauplii retained the ability to complete naupliar development but at a slower overall rate and with increased mortality relative to controls. These effects were more pronounced in the 96-h group. Increased mortality of stage VI nauplii was evident in both partially starved groups (7.1% for 48 h, 18.8% for 96 h) relative to unstarved controls (3.1%). Stage II nauplii exhibited little resistance to starvation and survival potential may have decreased as soon as 24 h.